Thrombopoietin (TPO) is the primary physiological regulator of thrombopoiesis, which is the formation of blood platelets. The first evidence for a humoral substance responsible for stimulating the production of platelets in thrombocytopenic rats was provided in 1958 (Keleman, E., et al., Acta Haematol. (1958) 20: 350-355). Since then, the purification and characterization of TPO, also referred to as megakaryocyte growth and development factor (MGDF) and c-Mpl ligand, have been achieved (de Sauvage, F. J., et al., Nature, (1994) 369: 533; Kuter, D., et al., Proc. Natl. Acad. Sci. USA (1994) 91: 11104; Sohma, Y., et al., FEBS Lett. (1994) 353: 57-61). The cloning and expression of TPO by standard techniques was also accomplished at approximately the same time (Bartley, T.D., et al., Cell (1994) 77: 1117; Lok, S., et al., Nature (1994) 369: 565), which later enabled the production of recombinant protein preparations for human clinical trials.
TPO is also one of the key cytokines driving the growth of stem cells and early hematopoietic progenitor cells. TPO alone stimulates clonal growth from single primitive CD34+CD38− human bone marrow cells, and it strongly synergizes with c-kit ligand (KL), flt3 ligand (FL) and IL-3 to enhance clonogenic growth (Borge, O. J., et al., Blood (1997) 90: 2282; Muench, M. O. and Barcena, A., Pediatr. Res. (2004) 55: 1050. TPO also enhanced clonogenic growth in response to KL+FL+IL-3+IL-6+EPO by as much as 80% and resulted in 40-fold expansion of multipotent progenitors following a 14-day incubation, further implicating a key role for this cytokine in stem cell growth and early hemopoiesis (Borge, O. J., et al., Blood (1997) 90: 2282). TPO enhances the growth and differentiation of human embryonic stem cells (Srivastava, A. S. et al. Stem Cells (2007) 25:1456). Stem cell-based approaches have demonstrated promising results in the treatment of several diseases, such as myocardial infarction, soft-tissue injury, heart failure, repair of atherosclerotic vessels and diseases of the central nervous system (Urbich, C., and S. Dimmeler (2004). Circ. Res. 95:343; Sylvester, K. G., and M. T. Longaker (2004) Arch. Surg. 139:93; Yoon, Y. S. et al. (2005) Biol Cell. 97:253; Martino, G., and S. Pluchino. (2006) Nat. Rev. Neurosci. 7:395).
Platelets are essential to the blood clotting process. If platelet production is inhibited or platelet levels become lower than levels that are considered to be normal, then the patient can develop thrombocytopenia, a potentially serious condition for which a TPO mimetic could provide significant relief. Serious consequences of thrombocytopenia include fatigue, bleeding, bruising, hemorrhage, and increased mortality as a result of related conditions. Thrombocytopenia can result from reduced production of platelets or increased breakdown of platelets via multiple different mechanisms. The leading causes of thrombocytopenia are cancer chemotherapy, surgery, bone marrow or stem cell transplantation, radiation injury or treatment, severe bacterial infections, chronic viral infection, systemic lupus erythematosus, rheumatoid arthritis, treatment with other drugs causing thrombocytopenia and a disorder known as immune thrombocytopenic purpura (also known as idiopathic thrombocytopenic purpura) (ITP) where the body's ability to produce and maintain an adequate supply of platelets is reduced for various reasons. Common drugs causing thrombocytopenia include heparin, quinidine, quinine, sulfa-containing antibiotics, some oral diabetes drugs, gold salts, rifampin and interferon-alpha.
The preferred acute treatment for severe thrombocytopenia, for example due to liver transplantation, is platelet transfusion, which can be costly, difficult, and associated with risks (Kuter, D. J., et al., Blood (2002) 100: 3457). Various therapeutics for thrombocytopenia that have either direct or indirect effects on platelet production are also known. For example, the first line of treatment for patients with ITP generally is the corticosteroid prednisone (Louwes, H. et al., Ann. Hematol. (2001) 80: 728). Prednisone is effective in approximately one-third of patients and is thought to reduce the amount of platelet-associated autoantibodies, which can participate in the clearing of platelets (Fujisama, et al., Blood (1993), 81: 2872), although this mechanism is not completely understood. Another therapeutic for ITP is intravenous immunoglobulin (IVIg), which is thought to modulate the production of antibodies and the activity of macrophages in removing platelets (Siragam, V. et al., Nat. Med. (2006) 12: 688-692), although the pharmacological mechanism of action of IVIg is also poorly understood. Patients who are refractory to the above treatments, such as prednisone and IVIg, may be considered for surgical removal of the spleen, which is a primary site of platelet sequestration. However, this procedure may have serious associated adverse events and is not considered as a cure for ITP. The cytokine growth factor, interleukin-11 (IL-11), is also approved for treating thrombocytopenia and has been shown to significantly increase platelet levels in adults following myelosuppressive chemotherapy. However, unlike TPO, IL-11 has pleiotropic effects and may be associated with serious adverse events such as peripheral edema and atrial arrhythmia (Gordon, M. S., et al., Blood (1996) 87: 3615).
Recombinant TPO preparations were available recently for clinical evaluation in certain indications, such as cancer chemotherapy. Two such products, PEG-rHu-MGDF (Basser, R. L. et al., Lancet (1996) 348: 1279) and rhTPO (Vadhan-Raj, S. et al., Ann. Intern. Med. (1997) 126: 673), act to promote platelet production directly by binding to the cell-surface TPO receptor. Certain cancer therapeutic regimens, including agents such as platinum drugs, paclitaxel, cyclophosphamide, doxorubicin, and ifosfamide, are associated with thrombocytopenia and frequently require dose modification and/or platelet transfusions to manage complications such as bleeding. The TPO products, PEG-rHu-MGDF and rh-TPO, both showed positive results in clinical trials by reducing chemotherapy-induced thrombocytopenia and in some cases reduced the need for platelet transfusions. However, PEG-rHu-MGDF was associated with production of neutralizing antibodies, which resulted in serious, treatment-resistant thrombocytopenia, thus raising concerns over safety in long-term administration. Both of the recombinant TPO products have been discontinued.
Recombinant TPO was also shown to be useful in the treatment of thrombocytopenia associated with human immunodeficiency virus (HIV) infection (Sundell, I. B., and Koka, P. S., Curr. HIV Res. (2006) 4: 107). During HIV infection, megakaryopoiesis, which is the process by which hematopoietic stem cells in the bone marrow differentiate into mature megakaryocytes, may become inhibited and the megakaryocytes may become infected with the virus, thus lowering the production of platelets. It is in this lowered state of platelet production that a TPO mimetic may be of therapeutic utility.
It is also known that healthy apheresis donors may require supportive treatment to increase platelet levels either prior to or after such procedures (Kuter, D. J. et al., Blood (2001) 96: 1339). A single injection of PEG-rHu-MGDF to healthy volunteers resulted in a dose-dependent increase in platelet counts and a correspondingly higher platelet yield from apheresis. This also suggests a potential use of a TPO mimetic compound.
It is known that thrombocytopenia can result from a disease or condition that results in reduced numbers of CD34+ human primary cells and further that such cells are responsive to recombinant TPO and TPO mimetics in animals, including humans, and in culture. This responsiveness results from the expression of TPO receptor on the surface of the cells and the intracellular signaling, including activation of STAT5, that is caused by binding of TPO or a mimetic to the receptor. Therefore, assays using these human primary cells in culture are particularly useful in evaluating the utility of novel TPO mimetics in animals, including human (Erickson-Miller, C. L., et al., Exp. Hematology (2005) 33:85-93). CD34+ cells are also known to differentiate into megakaryocytes in vitro and in vivo in response to recombinant TPO and TPO mimetics (de Sauvage, F. J., et al., Nature, (1994) 369: 533; Kuter, D., et al., Proc. Natl. Acad. Sci. USA (1994) 91: 11104; Erickson-Miller, C. L., et al., Exp. Hematol., (2005) 33:85). Recently, the International Application Publication Number WO2001/089457 disclosed synthetic TPO mimetic compounds for the treatment of thrombocytopenia. Such TPO mimetics have been shown to enhance platelet levels in humans in Phase I clinical trials, as described in the reference, Jenkins, J. M., et al., Blood (2007) 109:4739-4741, which illustrates that the utility of a TPO mimetic can be determined at an early stage compared to certain human therapeutics.
The present invention describes TPO mimetics that fulfill current needs and are useful as promoters of thrombopoiesis and megakaryopoiesis.